A rapid method for reconstitution of bacterial membrane proteins

A. Varadhachary, P. C. Maloney

Research output: Contribution to journalArticle

Abstract

We have devised a simple method for the reconstitution of bacterial membrane proteins directly from small (1-20ml) volumes of cell culture, thus eliminating the preparation of membrane vesicles. Cells are subjected to simultaneous lysozyme digestion and osmotic lysis, and after brief centrifugation ghosts are solubilized in 1.2% octyl-β-D-glucopyranoside (octyl-glucoside) in the presence of added carrier lipid and an osmolyte. Aliquots of the clarified supernatant are suitable for reconstitution, as documented by using extracts from three different Gram-negative cells to recover both inorganic phosphate (Pi)-linked antiport and oxalate:formate exchange activities in proteoliposomes. These proteoliposomes are physically stable, non-leaky and can sustain a membrane potential and, because functional porins do not reconstitute, the artificial system has transport characteristics similar to those found when proteoliposomes are obtained using standard methods. This method should become an important tool for the screening and characterization of large numbers of strains, both wild-type and mutant.

Original languageEnglish (US)
Pages (from-to)1407-1411
Number of pages5
JournalMolecular Microbiology
Volume4
Issue number7
StatePublished - Jul 1990

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Bacterial Proteins
Membrane Proteins
formic acid
Porins
Oxalates
Ion Transport
Muramidase
Centrifugation
Membrane Potentials
Digestion
Cell Culture Techniques
Phosphates
Lipids
Membranes
proteoliposomes

ASJC Scopus subject areas

  • Molecular Biology
  • Microbiology

Cite this

Varadhachary, A., & Maloney, P. C. (1990). A rapid method for reconstitution of bacterial membrane proteins. Molecular Microbiology, 4(7), 1407-1411.

A rapid method for reconstitution of bacterial membrane proteins. / Varadhachary, A.; Maloney, P. C.

In: Molecular Microbiology, Vol. 4, No. 7, 07.1990, p. 1407-1411.

Research output: Contribution to journalArticle

Varadhachary, A & Maloney, PC 1990, 'A rapid method for reconstitution of bacterial membrane proteins', Molecular Microbiology, vol. 4, no. 7, pp. 1407-1411.
Varadhachary, A. ; Maloney, P. C. / A rapid method for reconstitution of bacterial membrane proteins. In: Molecular Microbiology. 1990 ; Vol. 4, No. 7. pp. 1407-1411.
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